aadA as selection gene - big big problems (Nov/13/2007 )

Hello everybody!

I am trying to insert pCAMBIA1302 in Agrobacterium by electroporation. Controls transformed with bigger plasmid pBI121 use to grow well, while I still can't obtain a single transformed colony carrying pCAMBIA1302. I used the same stocks (bacteria, antibiotics, media...) for both control and experimental cultures and I really can't figure out what's exactly going on that's preventing my cells from being transformed/selected...

Anyway - after several tries - I began to suppose that there's something wrong with selection.pCAMBIA1302 carries aadA for amynoglicosides adenyltransferase as bacterial selection gene, and CAMBIA instructions say to perform colonies selection on kanamycin. As I browsed literature, I found references (Joung et al., 2000 - PNAS, Hollingshead & Vapnek, 1985 - PLASMID, Michael et al., 2006 - Microbes & infections) about the ability of that gene to confer resistance to aminoglycosides streptomycin and spectinomycin. On the contrary I couldn't find any specifical evidence about aadA as kanamycin resistance gene.Are you familiar with that gene? What is your experience in using it as bacterial selection marker?

Since my bacteria carry a streptomycin R gene on the helper, do you think it's reasonable if I make a try to electroporate cells again and select them on spectinomycin instead of kanamycin?

Any comment, help or hint will be of great importance.Thank you very much in advance,ILA

-ila-

You're posting in the wrong section -- repost in molecular biology or microbiology.

This is a very confusing area. The genes for kanamycin resistance, gentamicin, and neomycin are often mixed up, and present in bi-functional overlapping specificities. Additionally, the gene complexes often carry strep and spect resistance genes. I'd recommend that you debug using resistance in E. coli as a first step, and look very carefully at the literature (both on your plasmid, and on the gene). Also, don't trust what someone else says the gene is. Use the sequence, or, if necessary, sequence it yourself, to find the specific gene you are talking about. Plasmid gene names are often wrong.

-phage434-

QUOTE (ila @ Nov 13 2007, 06:29 AM)

Hello everybody!

I am trying to insert pCAMBIA1302 in Agrobacterium by electroporation. Controls transformed with bigger plasmid pBI121 use to grow well, while I still can't obtain a single transformed colony carrying pCAMBIA1302. I used the same stocks (bacteria, antibiotics, media...) for both control and experimental cultures and I really can't figure out what's exactly going on that's preventing my cells from being transformed/selected...

Anyway - after several tries - I began to suppose that there's something wrong with selection.pCAMBIA1302 carries aadA for amynoglicosides adenyltransferase as bacterial selection gene, and CAMBIA instructions say to perform colonies selection on kanamycin. As I browsed literature, I found references (Joung et al., 2000 - PNAS, Hollingshead & Vapnek, 1985 - PLASMID, Michael et al., 2006 - Microbes & infections) about the ability of that gene to confer resistance to aminoglycosides streptomycin and spectinomycin. On the contrary I couldn't find any specifical evidence about aadA as kanamycin resistance gene.Are you familiar with that gene? What is your experience in using it as bacterial selection marker?

Since my bacteria carry a streptomycin R gene on the helper, do you think it's reasonable if I make a try to electroporate cells again and select them on spectinomycin instead of kanamycin?

Any comment, help or hint will be of great importance.Thank you very much in advance,ILA

I've never used the CAMBIA vectors myself, but I know that they are commonly used. Hence, I would not question whether the antibiotic resistance is right. If you are trying to transform both a helper plasmid and cambia at the same time then you may be having difficult because you need both to enter the cell in order to get the right transformants. I'm guessing that pBI121 doesn't need a helper and therefore it is easier.

If this is the case, then you could either 1. let the cells grow a day longer or 2. transform the helper first, then make new electrocompetent agro that contain the helper and transform these with your cambia vector. I believe there are also cells already available that have Ti helper present in them. You might want to check into obtaining some these as it would save you time. If you are already using these and still aren't getting transformants then again, I would try to grow them a day longer. Make certain that you are doing a control transformation that doesn't contain any DNA so that you can distinquish what might be contaminating growth.

Also, you wouldn't want to select for just the helper by itself because then you won't be certain that the cells will contain your cambia vector. You'll need both in order for the vector to insert into plants.